Thailand

Country Brief Review On

Research and Development on Avian Influenza

November 29, 2005

co-host organizer write-up

- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development agency (NSTDA), Ministry of Science and Technology (MOST)

- Health Systems Research Institute (HSRI), Ministry of Public Health (MoPH)

Executive Summary

The avian influenza outbreak was witnessed in Thailand for the first time during the early of 2004. It has developed the tendency to become endemic, and more outbreaks are expected in the years to come. Moreover, the adaptive mutation or reassortment of the virulent avian influenza virus and the human influenza virus may lead to a more efficient form of transmission of the virus which could lead to human-to-human transmission of the virus, and thus triggering an influenza pandemic, which would take many lives. Due to its multidimensional nature, it is therefore necessary to seriously establish a regional research collaboration between Thai avian influenza research partnership (MoPH, NRCT, NSTDA, TRF, HSRI, NHF, and DLD) and four neighbouring countries (China, Indonesia, Vietnam, and Cambodia). The main objective of the collaboration is to strengthen national and regional research capacities to address pandemic influenza and to reduce national and regional coordination difficulties in the provision of public health to the sharing and exchange of information, ideas and practice. The International Development Research Center, Canada (IDRC) plans to support the regional research initiative with these countries.

To achieve a productive collaboration among the participating countries and the IDRC. All of Thai research institute agreed to join regional network to facilitate AI surveillance & control and also write-up the country brief review for the first intercountry partnership meeting. HSRI and NSTDA were assigned to be the co-host organizations for the brief review write-up. This review consists of 3 sections including an introduction on avian influenza situation and trends of influenza both on avian and human in the first section. The second section is the National Strategic Plan for Avian Influenza control in Thailand (in brief) and the last section is the proposed priority researches for collaboration with IDRC as followings;

1. Surveillance and disease control in birds (NRCT/TRF/DLD/DNP) a) Migratory/ wild birds/ non-industrialized poultry b) Vaccination in non-industrialized poultry c) Networking/ capacity building d) Monitoring of genetic variation/ evolution e) Effectiveness implications of disinfectant in outbreak

2. Husbandary model and alternative model/compartmentalization (NRCT/TRF/DLD)

3. Surveillance and disease control in humans (NSTDA/NHF) a) Asymptomatic infection b) Immune response in survivors

4. Protocol development for outbreak control at source of origin/ study control of host factors/ treatment of infected patients a) Monitoring genetic variation/ evolution b) Risk communication

5. Development of antiviral and vaccine (NSTDA) a) Oseltamivir/ zanamivir/ herb b) Vaccine development & clinical trial c) Antiviral resistance monitoring

6. Socio-economic: operational research on public health system of country members. (NSTDA/HSRI/TRF)

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Table of content

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Chapter 1 Introduction

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Chapter 2 Thailand Past Performance on Avian Influenza

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Chapter 3 National Strategic Plan for Avian Influenza Control in Thailand

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Chapter 4 Proposed Research for Collaboration with IDRC

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Chapter 1 Introduction

The recurrent outbreaks of avian influenza in 2004 and 2005 have become one of Thailand’s most exigent problems. Despite intensive efforts to contain the disease, not only does the problem remain unresolved, but its mounting adverse consequences have continued to wreck havoc on Thailand and many other countries. Evidently, avian influenza has evolved into a crisis situation partly because of inadequate knowledge and understanding about the disease.

Although scientific research and development in Thailand is of international standard, limited expertise in this newly emerging pathogen, as well as the complexity of the problem, has unfortunately hindered Thailand in its effort to solve it. It is thus crucial that Thailand acquire necessary knowledge to improve scientific and technological capability to the level that it can handle the crisis without depending solely on outside assistance. In doing so, networks of research and development, both among Thai scientific communities and overseas collaborators, must be promptly implemented.

More importantly, avian influenza has currently spread to almost all continents-- initially from Asia to Europe and recently to part of Africa and North America. This highly infectious disease has consequently become an enormous concern worldwide as it potentially leads to deadly global pandemic. To successfully control the disease, all countries, whether or not they are affected by the outbreaks, must act in concert to tackle this formidable threat.

Being among Southeast Asian countries frequently attacked by avian influenza, Thailand is well prepared for the surveillance and control of the disease. Nevertheless, to effectively restrain concurrent or upcoming epidemics, joint efforts from all countries in the region are, unquestionably, of paramount importance. As a result, the International Development Research Center (IDRC), located in Canada, has generously provided subvention to countries afflicted by avian influenza, namely, Thailand, Vietnam, Cambodia, Indonesia and China to augment collaborative research networks within the region and to foster more effective surveillance and disease control through a number of research and development projects involving both poultry and humans.

To expedite the establishment of mutual network among East Asian countries, Health Systems Research Institute (HSRI), on behalf of Thailand, is hosting the first international meeting between delegates from the four countries and IDRC. The conference will be held at Monthien Riverside Hotel, Bangkok, from November 30 to December 2, 2005. Participants of the meeting comprise experts from various government agencies appointed to solve avian influenza problems including Ministry of Public Health (Department of Medical Sciences and HSRI), Ministry of Agriculture and Cooperatives (Department of Livestock Development), Ministry of Science and Technology (National Center for Genetic Engineering and Biotechnology), and Office of Prime Minister (National Research Council of Thailand). The representatives from each country will present an overview of current situation and exchange their views on possible scenarios of avian influenza epidemics. In addition, ongoing research and development projects on human/avian influenza as well as future collaborative research will be discussed among the panel members.

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Chapter 2 Thailand Past Performance on Avian Influenza

1. National Strategy for Avian Influenza Epidemic Prevention

In an attempt to tackle the avian influenza problems, Deputy Prime Minister

Jaturon Chaisaeng, chairman of the national avian influenza committee, had initiated the avian influenza strategic committee to draft the National Strategic Plan for the Control of Avian Influenza and the National Strategic Plan for Influenza Pandemics Preparedness.

The five-year national strategic plan was approved by the cabinet on January 25, 2005, comprising 6 main areas as follows:

1. Development and management of the disease-free livestock system 2. Surveillance and disease control during an outbreak 3. Management of knowledge on the avian influenza virus 4. Potential enhancement of organizations and individuals 5. Better understanding about the disease and greater participation from the civic

and business sectors 6. Integrated management and the development of strategies to deal with the

disease Moreover, the Thailand National Strategic Plan for Influenza Pandemics

Preparedness has also simultaneously developed and approved at the same time. The plan comprise 5 strategies as follows:

1. Strengthening influenza surveillance systems 2. Preparedness of essential medical supplies and equipment 3. Preparedness for pandemic responses 4. Public relations and education 5. Development of sustainable and integrated management systems

2. Avian Influenza Research Accomplishment

Since the outbreak of avian influenza in Thailand, the Thai government has funded several R&D projects through various agencies. Some examples of the accomplishment are following. 2.1 “Avian Influenza Rapid Diagnostic Kit”

Thai scientists have successfully developed the influenza rapid diagnostic kit. Not only can this technology be used to effectively diagnose the H5N1 strain of avian influenza virus, but it also requires as little as 10 minutes to complete the test. The kits are now available for commercial use from several companies in Thailand.

To develop rapid diagnostic tests specific to subtypes of hemagglutinin, a project to produce recombinant H1, H3, H5, H7, H9, and nucleoprotein as well as their corresponding monoclonal antibodies has been funded.

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Within six months after the project was initiated, the research team was able to screen and identify antigenic epitopes, which effectively boost the immune response, for hybridoma preparation. The study indicated that recombinant antigens derived from H1, H3 and H5 influenza viruses could elicit a significant level of antibody response in immunized mice. To date, twelve clones of H1 hybridoma have been made and those of H3 and H5 are under development. All

clones, once completely generated, will be evaluated for their specificity for hemagglutinin (H) or nucleoprotein (N). 2.2 “Molecular Biology-based Detection of Avian Influenza Virus”

Methods for avian influenza H5N1

detection in the laboratory using PCR and RT-PCR have been developed. Both methods are now patented and available for research use at BioService Unit of BIOTEC. Moreover, the kits will soon be available commercially.

Since, besides avian influenza

H5N1 virus, other pathogenic strains of avian influenza A virus, such as H7N7 and H9N2, have been known to transmit directly from animals to humans, projects aiming to develop detection methods for avian influenza virus subtype H5, H7 and H9 using molecular biology approach has been funded. Expectedly, this technology will serve as a powerful tool for epidemic surveillance as it can rapidly and accurately detect all three subtypes of avian influenza virus in infected animals. Moreover, the methods are currently being tested for their compatibility to use with human samples.

2.3 “Epidemiological Studies”

Avian influenza viruses isolated from both infected animals and humans has been subjected to genomic sequencing. The results revealed that the avian influenza virus isolated in Thailand resembled those of Hong Kong and Vietnam, which was the strain originally isolated in China in January 2002. Notably, the study has provided more insights into better understanding of not only

characteristic of the virus, but also its epidemiology and ways to monitor possible virus reassortment. These breakthrough findings had prompted Thai investigators and their international collaborators to publish an article in the July 8, 2004 issue of Nature, entitled “Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asian. Another study was reported in the October 25, 2004 issue of Virology

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demonstrating that eight pairs of primers that are specific for all eight genes of avian H5N1 influenza virus could be employed to detect virus reassortment in both animals and humans. Thanks to this study, monitoring new strains of H5N1, which potentially can be transmitted from human to human, was not only possible but easily accessible.

This technique was first used to determine whether there existed a human-to-human transmission in H5N1-infected mother and child in Kumpaengpetch province. While tissue samples could not be collected from the deceased child, tissues from various organs of the mother, who never had close contact with infected chickens, could be obtained. Since the samples were formalin-treated, they could not be amplified by the eight primer-based PCR assay. To circumvent the problem, the researchers changed the approach to sequence specifically at the receptor-binding site as well as fragments of other genes to determine whether reassortment between human and avian influenza viruses had occurred. The results indicated that there was no change at the receptor-binding site of the HA, and the nucleotide sequences of all eight genes were closely related to the strain of the H5N1 virus isolated in Thailand, thereby suggesting that reassortment had not yet occurred. However, given that the mother had a close contact with her sick child but no recognized exposure to poultry, the researchers could not rule out the possibility of human-to-human transmission. Unfortunately, further investigation could not be performed as the bodies of infected individuals were already cremated. Detailed results of this study were reported in New England Journal of Medicine 2005 Jan 27; 352(4):333-40. Epub 2005 Jan 24. Probable person-to-person transmission of avian influenza A (H5N1). Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, Puthavathana P, Uiprasertkul M, Boonnak K, Pittayawonganon C, Cox NJ, Zaki SR, Thawatsupha P, Chittaganpitch M, Khontong R, Simmerman JM, Chunsutthiwat S. The investigators from faculty of Veterinary Science, Chulalongkorn University have studied the molecular epidemiology of avian influenza H5N1 viruses isolated in Thailand by monitoring changes in amino acids in protein regions known to affect pathogenicity of the virus. Nucleotide sequencing of 4 genes including HA, NA, M and NS were performed in 40 samples of various animal species and genetic comparison of H5N1 influenza viruses isolated from each outbreak site was analyzed. The results indicated that viruses isolated during recent outbreaks in Suphanburi from June-July 2005 showed no signs of virus reassortment. Moreover, the avian influenza H5N1 isolated in Thailand belonged to genotype Z, which was genetically closely related to the strains isolated in Vietnam, but not those isolated in Indonesia, Hong Kong and China. In addition, the investigators could detect avian influenza virus in other avian species such as wild birds, crows, peacocks, pheasants, and pigeons. Nucleotide sequences of avian influenza isolated in Thailand were already submitted to GenBank database (http://www.ncbi.nlm.nih.gov), which will be useful for research involving detailed analysis of avian influenza virus and for surveillance and control of the disease.

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The researchers from National Institute of Animal Health, Department of Livestock Development, Ministry of Agriculture has investigate of avian influenza viruses in various non-avian species mammals such as dogs, cats, pigs and other domesticated animals in frequently occurring outbreak areas. The results obtained from this study not only served as epidemiologic data for future avian influenza H5N1 virus studies in mammals, but also provided insights into the prevention and eradication of avian influenza in Thailand. As target areas, the investigators have selected villages that have history of sick or dead people as well as those in which outbreaks in poultry frequently occurred. According to the results from 1,234 samples obtained from 33 areas in 12 provinces, no avian influenza virus could be detected. Moreover, samples from geese, cows and goats were seronegative for H5N1 virus, whereas those of local chickens and ducks were seropositive with 0.7% and 56.5%, respectively. The study also found that some samples obtained from dogs and cats might be reactors, which need to be further verified by Western blot analysis.

Because of frequent avian influenza outbreaks in Thailand since January 2003 and of several reports from Japan and Korea suggesting that migratory birds, especially those in the genus Anatidae, might be the primary reservoir of the avian influenza virus, the investigators from faculty of Natural Resources, Prince of Songkla University have conducted a preliminary study to detect infection of avian influenza viruses subtype H5 on a variety of bird species in Southern Thailand. Samples of birds residing in mangrove forests or swamp areas in 5 southern provinces, namely, Phuket, Trung, Pattani, Songkla and Nakhonsrithammarat were collected from December 2004 to May 2005. Of the total 160 samples, 132 samples were from local birds and 28 samples were from 7 species of migratory birds. Viruses were isolated and propagated in chicken embryonic eggs. Haemagglutination (HA) and Haemagglutination inhibition assays were also performed with all samples. The results revealed that no avian influenza viruses subtype H5 could be detected. However, due to small sample size and no samples from genus Anatidae, it was inconclusive as to whether or not wild birds carried the virus. Despite there being no concrete evidence, surveillance and sample collection should still be performed routinely.

From the collaborative effort of Faculty of veterinary medicine, Faculty of medicine, Faculty of agriculture, Faculty of economics, Faculty of social sciences of Chiang Mai University and Department of Livestock Development. They were established the research project entitled “Evaluation of outbreak impacts, recovery measures, surveillance and control of avian influenza in northern Thailand” The aims of the project are to evaluate socio-economic impacts as a result of avian influenza outbreaks and to study epidemiology of avian influenza using data obtained from statistical analyses, laboratory investigation and geographical information system to help local farmers and staff exercise appropriate surveillance. The study revealed that the government’s measures to contain avian influenza outbreak had a significant impact on farmers. Some of the poultry farmers have changed their career, thereby directly affecting the lifestyle of the local people as the loss of income lead to accumulating household debts. During the first outbreak, poultry consuming was believed to have a high risk of contracting the disease, resulting in a progressive decline in poultry consumption, which widely affected farmers as well as those involved in poultry business. Moreover, most of the troubled farmers were unable to negotiate a loan from commercial banks for their financial burdens. The investigators also found that avian

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influenza is likely an endemic disease as suggested by immune response in chickens that had never been immunized. Furthermore, common risk factors include transferring infected animals into the area, having insufficient biosafety, sharing water supplies. It is recommended that development of geographical information system covering the entire northern region should be routinely conducted. Moreover, surveillance network, development of avian influenza diagnostic kits, characterization of virus, and improvement of poultry slaughterhouse and cock-fighting field should be taken into account for future research.

The research project entitled “Epidemiologic investigation of avian influenza in

avian species found in central and western Thailand” was a collaborative work of Kasetsart University by Faculty of veterinary medicine, Suwanvajukkasikij Institute and Department of Livestock Development, aiming to develop the surveillance network for avian influenza in Nakhonpathom and Supanburi provinces. The study was conducted through a survey of general knowledge and possible risk factors of avian influenza. In addition, the study was focused on applying epidemiology of avian influenza in chickens in the Geographical Information System (GIS) for outbreak surveillance. The study revealed that, during the first outbreak, most of the affected farmers were those with small farm, who had a high risk of contracting the disease because of poor hygienic practices such as cleaning cages without germicide, letting poultry wander in open land, using no protection when in close contact with animals. For the development of GIS, the system includes information of poultry number raised within the two provinces. The system also can be used to seclude suspected birds in case of an outbreak. Moreover, it is a useful tool to estimate the cost of damage to be compensated to the affected farmers. To develop the surveillance network for avian influenza, four strategies should be implemented as follows: 1) brainstorm meeting of those exercising surveillance from both civic and private sectors as well as from local residents 2) workshop activities on control and prevention of disease 3) situation model and 4) analytical interview. As a consequence of this study, surveillance network system among different sectors was established. 2.4 “Pathogenesis of avian influenza virus (H5N1) infection in Humans” While infection of avian influenza virus in primates such as monkeys and humans is confined mostly to the upper respiratory tract, infection in poultry and other mammals such as mice, cats and tigers is widely disseminated. To date, the mechanisms underlying difference in infection in primates and other mammals are not clearly understood. It has been postulated, however, that differences in sialic-acid containing receptor expression might play a pivotal role in determining tissue tropism of the virus. To address this issue, a research team at Siriraj Hospital performed an autopsy on a patient who died from avian influenza and, subsequently, carried out a thorough virologic diagnosis in various tissue samples using RT-PCR and immunohistochemistry. The results demonstrated that H5N1 virus replication sites in humans were restricted to lung and intestine, which was distinct from those found in other infected animals. Moreover, the study also identified type II pneumocytes in the

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lung as the major site of H5N1 virus replication in humans. This study was published in EMERGING INFECTIOUS DISEASES Volume 11, No. 7, July 2005, 1036-1044. Influenza A H5N1 Replication Site in Humans. Mongkol Uiprasertkul, Pilaipan Puthavathana, Kantima Sangsiriwut, Phisanu Pooruk, Kanittar Srisook, Malik Peiris, John M. Nicholls, Kulkanya Chokephaibulkit, Nirun Vanprapar, and Prasert Auewarakul. 2.5 “Research and Development of Anti- influenza Drugs” Virtual Screening from Thai Medicinal Herbs

A project aiming to screen for compounds that potentially could inhibit neuraminidase enzymatic activity has been launched. Based on the database of bioactive compounds isolated from natural products such as Thai medicinal herbs and on the molecular database consisting of 2,684 compounds, the team identified 18 structures that showed promising potential. These compounds are currently being evaluated for their ability to inhibit influenza virus replication by comparing their efficacy to standard anti-viral drugs such as Oseltamivir or TamiFlu. The results obtained from this study will be valuable for future development of traditional Thai medicinal

herbs as an alternative for imported anti-virus drugs, which may be in shortage in case of influenza pandemic. The research team also endeavors to screen more neuraminidase inhibitors from database of bioactive compounds isolated from natural products and molecular database of 50,000 compounds using a comprehensive molecular docking technique, which is expected to identify more candidates to be tested for their efficacy.

Preliminary results have demonstrated that the virtual screening technique used

in phase 1 was relatively practicable compared to results obtained from laboratory experiments. This technology has been submitted to Comb.Chem. High Through. Screen. 2005 and J. Chem. Inf. Comput. Sci. entitled “The Combination of Molecular Docking and Kohonen Neural Networks as a Novel Virtual Screening Tool”.

Synthesis of Oseltamivir If influenza pandemic occurs, the supplement of anti-viral drug will be limited to

only high-income country. For country’s security, Thailand aims to develop a capability for Oseltamivir production. Since currently available route for Oseltamivir synthesis is relatively long and complicate, the research team is now developing an alternative synthesis route which is expected to be used in pilot-scaled production with more ease. Fortunately, the 6 out of 12 steps have been accomplished and the yields of synthesis are well acceptable. More than 85-90% of products of each step could be harvested.

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2.6 “Research and Development of Avian Influenza Vaccines”

Recombinant Protein-based Prototype Vaccine A recombinant protein-based prototype vaccine against avian influenza has been

developed. The research team has generated recombinant antigenic proteins, including HA, NA, M, and NS, from H5N1 virus isolated in Thailand to boost humoral immune response in immunized subjects. All four proteins have been successfully engineered and tested for their immune response in mice. The results revealed that all proteins were safe and able to elicit immune response in immunized mice. The next step is to assess the vaccine efficacy in poultry. Nevertheless, to achieve this goal, Thailand needs to set up laboratories equipped with high level of biosafety (BSL3+). These facilities will be essential not only for research involving infectious diseases, but also for upgrading and strengthening the country’s research capability.

Currently, a plan for BSL3+ animal research facility is being developed. The project is expected to be started in mid 2006.

Reverse Genetics Prototype Vaccine A reverse genetics H5N1 strain has already been

constructed and will be used for vaccine production research. This technology will assist scientists to design and develop vaccines in a timely manner especially in case of the epidemic of new strains of virus.

Pandemic Influenza Vaccine As the World Health Organization (WHO) has issued a dramatic warning that

avian influenza will trigger an international pandemic that could cause a potential global crisis, it is necessary that effective prevention as well as facilities for a large-scale vaccine production should be promptly implemented.

To prepare for a potential influenza pandemic, Department of

Disease Control and Department of Medical Sciences, has formed a pandemic influenza vaccine committee, chaired by Dr. Paijit Warachit, Director of Department of Medical Sciences. One of the primary goals of the committee is to establish a large-scale vaccine manufacturing facilities and to procure appropriate technology by negotiating with companies\countries that are capable of transferring technology necessary for a vaccine production to be used during seasonal outbreaks. This technology will be fundamental to a large-scale vaccine production in preparation for a potential influenza pandemic. The committee is also in the process of finding possibility to manufacture and test pandemic vaccine in volunteers. Moreover, reverse genetic vaccine and recombinant genetic vaccine will soon be in clinical trials. Importantly, the committee is drafting a master plan to promote pandemic vaccine manufacturing and outlining the influenza vaccine policy that conforms to the National

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Vaccine Plan. With regard to research and vaccine development project directed by The Ministry of Public Health, according to the preliminary meeting, the ministry agreed to collaborate with the Chinese government to launch a number of vaccine projects.

3. Knowledge management The program is set up to make sure that scientific knowledge relating to avian influenza and emerging diseases is used for prevention and control of the diseases in a rational and timely fashion. At its current early stage, a few areas have been focused.

1. Policy options and recommendations. The program initiates or facilitates discussion on difficult policy or strategic issues among relating institutes and other stake holders with the aims to generate options of recommendations for government or other institutions. The topics of current interest includes the strategic uses of influenza vaccines in avian species and the national investment on human vaccine research, development and manufacturing.

2. International collaboration. The program initiates and maintains scientific communication among Thai scientists and between Thai and foreign scientists. Recent activities include a national scientific meeting on avian influenza and the collaborative visit to Harbin Veterinary Research Institute as well as organization of visit of foreign scientists to Thailand.

3. Public communication. The preparation against pandemic influenza requires massive mobilization of various kinds of resources and, therefore, requires the participation of a large number of people as well as strong support from public. The program aims to disseminate the appropriate level of knowledge to public through various media, including Internet and press. Several activities with press have been planned such as press education (providing appropriate scientific training to press personnel), press update (making up-to-date data available to press) and press conference.

4. Publications in International journals 1. Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS; Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia., Nature 2004 Jul 8;430(6996):209-13 2. Kumnuan Ungchusak , Prasert Auewarakul, Scott F. Dowell; Rungrueng Kittpathi, Wattana Auwanit, Pilaipan Puthavathana, Mongkol Uiprasertkul, Kobporn Boonnak, Chakrarat Pittayawonganon, Nancy J. Cox, Sherif Zaki, Pranee Thawatsupha, Malinee Chittaganpitch, James M. Simmerman, Supamit Chunsutthiwat. Probable

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Person-to-Person Transmission of Avian Influenza A (H5N1), Thailand. Submitted to New England Journal of Medicine 2005. N Engl J Med. 2005 Jan 27;352(4):333-40. Epub 2005 Jan 24. 3. Juthatip Keawcharoen, Kanisak Oraveerakul, Thijs Kuiken, Ron A.M. Fouchier, Alongkorn Amonsin, Sunchai Payungporn, Suwanna Noppornpanth, Sumitra Wattanodorn, Apiradee Theamboonlers, Rachod Tantilertcharoen, Rattapan Pattanarangsan, Nlin Arya, Parntep Ratanakorn, Albert D.M.E.Osterhaus and Yong Poovorawan; 2004. Avian Influenza A (H5N1) virus fatal for tigers and leopards; Emerging Infectious Diseases. 2004;10:2189-2191 4. Uiprasertkul, M., Puthavathana, P., Sangsiriwut, K., Pooruk, P., Srisook, K., Peiris, M., Nicholls, J.M., Chokephaibulkit, K., Vanprapar, N. and Auewarakul, P. (2005). Influenza A H5N1 Replication sites in humans. Emerging Infectious Disease, 2005 11(7), 1036-1039.) 5. Sunchai Payungporn, Piraya Phakdeewirot, Salin Chutinimitkul, Apiradee Theamboonlers, Juthatip Keawcharoen, Kanisak Oraveerakul, Alongkorn Amonsin and Yong Poovorawan; 2004. Single step multiplex RT-PCR for influenza A virus subtype H5N1 detection; Viral Immunology, 2004;17:588-593 6. Nareerat Viseshakul, Roongroje Thanawongnuwech, Alongkorn Amonsin, Sanipa Suradhat, Sunchai Payungporn, Juthatip Keawchareon, Kanisak Oraveerakul, Piya Wongyanin, Sukanya Plitkul, Apiradee Theamboonlers, Yong Poovorawan; The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand. Virology. 2004; 329:169-176 7. Pilaipan Puthavathana, Prasert Auewarakul, Pakapak Chor Charoenying, Kantima Sangsiriwut, Phisanu Pooruk, Kobporn Boonnak, Raweewan Khanyok, Pranee Thawachsupa, Rungrueng Kijphati, and Pathom Sawanpanyalert. Molecular characterization of complete genome of human influenza H5N1 virus isolates from Thailand Journal of General Virology (JGV) J Gen Virol. 2005 Feb;86(Pt 2):423-433 8. Sabgnam C.: Chuakheaw, D.; Sanbandit, K.; Jongkol, N.; Jongkol, N.; Nunream, P.; Hannongbua, S., “The Combination of Molecular Docking and Kohonen Neural Networks as a Novel Virtual Screening Tool”,- J. Chem. Inf. Comput. Sci.- Combinatorial Chemistry & High Throughput Screening. 9. Sunchai Payungporn, Salin Chutinimitkul, Arunee Chaisingh, Sudarat Dumrongwantanapokin, Chatanee Buranathai, Alongkorn Amonsin, Apiradee Themboonlers, Yong Poovorawan. Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. J. Virol. Methods., 2005 10. Thanawongnuwech R, Amonsin A, Tantilertcharoen R, Damrongwatanapokin S, Theamboonlers A, Payungporn S, Nanthapornphiphat K, Ratanamungklanon S, Tunak E, Songserm T, Vivatthanavanich V, Lekdumrongsak T, Kesdangsakonwut S, Tunhikorn S, Poovorawan Y. Probable tiger-to-tiger transmission of avian influenza H5N1. Emerg Infect Dis. 2005 May;11(5):699-701. Emerg Infect Dis. 2005 Jun;11(6):976. 10. Amonsin A, Payungporn S, Theamboonlers A, Thanawongnuwech R, Suradhat S, Pariyothorn N, Tantilertcharoen R, Damrongwantanapokin S, Buranathai C, Chaisingh A, Songserm T, Poovorawan Y. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology. 2005 Sep 26; 11. Payungporn S, Tangkijvanich P, Jantaradsamee P, Theamboonlers A, Poovorawan Y. Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis. J Virol Methods. 2004 Sep 15;120(2):131-40. 12. Juthatip Keawcharoen, Kanisak Oraveerakul, Sumitra Wattanodorn, Alongkorn Amonsin, Termsitthi Papravasit, Somkid Karnda, Karn Lekagul, Rattapan Pattanarangsan, Suwanna Noppornpanth, Ron A.M. Fouchier, Thijs Kuiden, Albert

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D.M.E. Osterhaus, Sanchai Payungporn, Apiradee Theamboonlers, Yong Poovorawan. Characterization of the hemagglutinin and neuraminidase gene of recent H5N1 avian influenza viruses among different avian species in Thailand. Acta Virologica. 2004 (revised) 5. Patents

Nucleotide sequence of Avian Influenza virus subtype H5N1 for virus detection and diagnosis in Thailand (Professor Dr. Yong Poovorawan’s project)

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Chapter 3

National Strategic Plan for Avian Influenza Control and Influenza Pandemic Preparedness in Thailand

Executive Summary BE 2548-2550 (A.D. 2005-2007)

The avian influenza outbreak was witnessed in Thailand for the first time during

the early of 2004. It has developed the tendency to become endemic, and more outbreaks are expected in the years to come. If no efficient control measures are put in place, and adaptive mutation or re-assortment of the virulent avian influenza virus and the common influenza virus that effect humans is observed, it could lead to a more efficient form of transmission of the virus which could lead to human-to-human transmission of the virus, and thus triggering an influenza pandemic, which would take many lives.

Due to its multidimensional nature, it is therefore necessary to seriously implement a strategic plan and formulate sustained systematic multi-stakeholder actions to counter the avian influenza problem. National Strategic Plan for Avian Influenza Control The plan, which was approved by the cabinet on January 25th 2005, has the following targets and operational strategies: Targets for the 3 years period (B.E. 2548-2550) (A.D. 2005-2007)

1. Targets for the control of spread in poultry 1.1 No spread of avian influenza in economic poultry within 2 years 1.2 Reduce the spread of the disease, so that avian influenza is not a

problem with domestic poultry, fighting cocks, exotic birds and migratory birds within 3 years.

1.3 No spread of avian influenza in other animals within 3 years

2. Targets for the control of spread in humans 2.1 No spread from animals to humans within 2 years 2.2 Efficient pandemic preparedness within 1 year

Strategies: There are 6 inter related strategies in the National Strategic Plan for Avian Influenza Control, which include: Strategy 1. Development of a disease free poultry management system. The objective is to enable poultry to be free from diseases and safe for consumption, base on the following measures: 1. Improve the structure and system for raising domestic poultry, exotic birds, fighting cocks and free range ducks, in accordance to the bio security standards. Identity cards should be issued for fighting cocks, particularly during movements.

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2. Develop zoning and compartment system, to clearly define areas for the movement control of poultry. 3. Develop efficient disease surveillance and response teams for poultry, to cover all areas. 4. Surveillance and control of avian influenza in natural birds. 5. Analyze the situation and prepare for use of vaccines for prevention of the disease in poultry. 6. Continuous publicity and provision of information and knowledge to the public.

Strategy 2. Disease surveillance and response during outbreaks. The objectives are to rapidly detect and control outbreaks of avian influenza in poultry and to closely follow up on the changes in the situation, base on the following measures:

1. Disease surveillance and control in animals, by implementation of proactive surveillance measures that would report back on disease outbreak within 12 hours, destroy the sick animals and their carcasses, disinfect the farms, restrict movement and consider appropriate use of vaccines.

2. Disease surveillance and control in humans by preparation for continuous active surveillance system, stockpiling of antivirals and influenza vaccines.

3. Influenza pandemic preparedness plan. 4. Set up ad hoc integrated mechanisms at all levels, to function as the

command centre during outbreaks, in order to bring about rapid control of the disease.

Strategy 3. Knowledge generation and management. The objective is to generate and manage knowledge which can be used to efficiently solve problems of avian influenza, base on the following measures:

1. Generate basic knowledge that is necessary for prevention and control of avian influenza in humans and animals.

2. Research and Development of vaccines which are ready for use during outbreaks, in humans and animals.

3. Research and Development on diagnostic kits which could lead to rapid screening.

4. Research and Development on medicines for treatment of avian influenza. 5. Establish a neutral technical body responsible for the management of

knowledge, which employs full time permanent staff. Strategy 4. Capacity building of organizations and manpower. The objective is to strengthen capacity building of organizations and manpower working on surveillance and control at all levels, base on the following measures:

1. Develop the capacity of disease surveillance and control in humans, with the establishment of efficient epidemiology units in every district, in addition to training of professional field epidemiologists and experts in other fields.

2. Develop diagnostic capacity in humans. Develop a system of network of laboratories at the central, provincial and hospital levels to support prompt and accurate diagnosis of avian influenza in humans.

3. Control of avian influenza in hospitals, by development of medical personnel in areas of infectious diseases, and establish isolation rooms for patients infected or suspected of being infected with serious communicable diseases.

16

4. Develop disease surveillance and control capacity in animals, by training and development, and provision of incentives for professional field veterinary epidemiologists, and promotion of the role of wild life conservationists.

5. Develop the capacity of laboratories for diagnosis of avian influenza in animals, with high safety levels.

Strategy 5. Create understanding and participation of the civil society and

private sectors. The objective is to strengthen the roles of civil society and private sectors, in prevention and control of disease outbreaks, base on the following measures:

1. Promotion of self grouping, for coordination between the small scale farmers and those in the concerned occupation.

2. Develop network of volunteers to monitor and control disease outbreak in the community.

3. Develop effective public communications at all levels.

Strategy 6. Develop sustainable integrated management systems and mechanisms. The objective is to create efficient and united mechanisms for the management of avian influenza problems, base on the following measures:

1. Develop mechanisms to manage problems of avian influenza under normal circumstances, by establishment of knowledge management mechanisms, whereby full time staff are employed under a permanent setting.

2. Develop mechanisms to manage problems of avian influenza during outbreaks, by establishment of a national level committee and a national level operations centre and develop management systems at local levels.

National Strategic Plan on Influenza Pandemic Preparedness.

Targets for the 3 years period (B.E. 2548-2550) (A.D. 2005-2007) To strengthen an effective influenza surveillance system.

To enable Thailand to be ready for the efficient management of the emergency situations during the influenza pandemic

To stockpile antiviral drugs (Oseltamivir) for treatment and to stockpile raw materials for the manufacturing of antiviral drugs (Oseltamivir)

To develop the capacity to manufacture / stockpile influenza vaccines

In case of an influenza pandemic, hospitals throughout the country have the capacity of up to beds for taking care of influenza patients in critical conditions. In case of outbreaks in specific areas, field hospitals will be ready for services.

Strategies: There are 5 strategies in the National Strategic Plan on Influenza Pandemic Preparedness, as follow:

Strategy 1 : Strengthening influenza surveillance systems: 1. Accelerate and intensify the surveillance systems in both animals and

humans. 2. Systematically link the surveillance information between the animals and

humans surveillance system.

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3. Strengthen the capacity of influenza surveillance networks in humans, including the community networks, the medical profession networks, the hospital networks and the laboratory networks.

Strategy 2 : Preparedness of essential medical supplies and equipment:

1. Stockpile adequate quantity of essential medical supplies, diagnostic test kits, and the necessary personal protection equipments, for use when necessary.

2. Develop efficient stockpiling and storage systems for drugs, vaccines and medical supplies.

3. Support research, development and production of vaccines and antiviral drugs, in order to be self dependent in the long run.

4. Set up criteria for the fair distribution of the limited available medical supplies, antiviral drugs and vaccines.

Strategy 3 : Preparedness for pandemic responses

1. Set up standard operating procedures for all organizations. 2. Develop the capacity of medical and public health officials, and various groups of

volunteers to be ready to provide appropriate care. 3. Strengthen the readiness and capacity of hospitals and prepare plans for patient care

systems during pandemic.

4. Develop public health emergency plans and measures.

5. Develop financial support system for activities considered necessary / urgent.

Strategy 4 : Public relations and education

1. Publicity of information to create knowledge and understanding on prevention and care of influenza.

2. Develop risk communication skills, for concerned officials.

3. Set up working groups comprising of all concerned sectors to officially communicate and coordinate on the provision of information to the public.

4. Formulate a communications plan, with the use of public communications tools at the national and local levels, in addition to establishment of networks with the media during pandemic.

Strategy 5 : Development of sustainable and integrated management systems

1. Develop mechanisms for influenza knowledge management, during the Pandemic Alert period.

2. Develop efficient mechanisms to effectively manage the situation during pandemic.

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Summary of Strategic Plan for Avian Influenza Control (A.D. 2005-2007)

3. Objectives: • Reduce

morbidity/mortality • Reduce economic/social

consequences • Regain confidence

5 Principles • Public safety • Eq

a• Biodiversity • E

6 Strategies/25 Measures

6. Sustain

• Sustaicondit

• surveillance/control/diagnosis in humand in animals

• disease treatment/control in hospitals

Support strategies

3. Knowledge generation/management (325million)

• Research and Develof vaccines / diagnmedicines

• Basic knowledge • Central knowledge

management body

5 Targets: Humans • No human infection

in 2 years • Pandemic

preparedness plan in1 year

uity in business nd lifestyle

• To

mphasis on important/urge

ability management syste

nable measures during normions and during outbreaks

1. Dman•

• •

• • •

2. O(1,5

•

•

•

•

ans/

opment ostic kits/

Mai

Animals • No outbreak in economic

poultry in 2 years • Not a problem to other poultry

in 3 years • No outbreak in other animals

in 3 years

hai context on the basis f knowledge

nt issues/results

m

al

isease free poultry agement (700 million baht) Raise according to bio security standards

Area zoning, mobility control Develop disease surveillance/control systems

Surveillance in natural birds Prepare for vaccine use Public relations

utbreak management 60 million)

n strategies

5. Social/private participation (240 million)

Surveillance/control in animals

Surveillance/control in humans

Specific mechanisms at all levels

• Network of disease surveillance and control volunteers

• Grouping of farmers • Communications/ Public relations

4. Capacity of organizations/manpower (1,201 million)

Pandemic preparedness plan

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Summary of the National Strategic Plan on Influenza Pandemic Preparedness 3 objectives and 5 targets

3 Objectives • Pandemic prevention • Reduction of morbidity and

mortality • Development of effective

respond systems

5 Targets • Surveillance system • Pandemic response • Stockpile of supplies and

drugs • Develop vaccine production

capacity • Public health service

system

5 Strategies and 18 measures:

4. Pubic relations and education • Information dissemination • Develop risk communication skills • Set up multi sectoral working groups • Formulate communication strategies

3. Pandemic Response Preparedness • Set up standard operating procedures • Develop the capacity of staff /

volunteers • Develop preparedness of hospitals • Develop capacity of public health

emergency measures • Develop financial measures

2. Stockpile of supplies • sufficient antiviral drugs for

emergency situation • Develop system for stockpiling and

administration of the stock • Research and development on

vaccines and antivirals • Develop criteria for fair distribution

1. Surveillance systems • Surveillance systems in humans and

animals • Link surveillance information of

humans and animals • Surveillance network of human

influenza

5. Sustainable and integrated management systems

• Pandemic alert period • Pandemic period

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Summary of R&D Operation Plan for Avian Influenza Control (A.D. 2005-2009)

Avian Influenza-Related Activities (5 Years Plan)

1. Research and development of diagnostic kits

1.1 Development and production of all subtypes (H1-H9) of human and avian influenza virus proteins 1.2 Generation of monoclonal antibodies against H1, H3, H5, H7 and H9 proteins

1.3 Manufacture of avian influenza rapid diagnostic kits

1.4 Manufacture of molecular biology-based diagnostic kits that detect all subtypes of avian influenza viruses 2. Research and development of human and poultry vaccines against avian influenza

2.1 Research and development of 10 alternative recombinant vaccines

2.2 Research and development of reverse genetic vaccines

2.3 Evaluation of immunogenicity of selected avian influenza antigens in mice

2.4 Efficacy testing of alternative vaccines in poultry. (Since there is currently no BSL3 facility for laboratory animals in Thailand, the project is to be conducted by overseas collaborators.) 2.5 Studies on avian influenza vaccine delivery systems 2.6 Evaluation of pandemic influenza vaccine efficacy

2.7 Preparation of a large-scale vaccine production by collaborating with Department of Medical Sciences in building fundamental structures and conducting feasibility studies both in technical and economic aspects 3. Research and development of anti-avian influenza drugs

3.1 Bioinformatics-based screening for bioactive compounds from Thai medicinal herbs and database of 50,000 chemical structures 3.2 Approximately 50 chemical structures showed promising potential as evaluated by enzymatic and cell-based assays 3.3 Among selected structures, approximately 10 candidates have been chemically modified to improve potency and reduce toxicity to be developed as antiviral drugs.

3.4 Experiments in laboratory animals

3.5 Synthesis and development of Oseltamivir

3.6 Oseltamivir production in a semi-industrial scale

3.7 Studies avian influenza viruses resistance on Oseltamivir

4. Epidemiologic studies

4.1 Genetic sequencing of avian influenza viruses isolated from infected humans

4.2 Genetic sequencing of avian influenza viruses isolated from birds and other animals, both wild and domestic 4.3 Studies on immune response of personnel who have contacted with avian influenza-infected patients 21

4.4 Research on probability of cross-species transmission of avian influenza using molecular biology techniques 4.5 Pathological studies using samples obtained from avian influenza infected animals and humans 5. Fundamental knowledge on avian influenza pathogenesis

5.1 Studies on avian influenza pathogenesis in fighting cocks and/or open-farm chickens to predict time frame and kinetics of disease transmission 6. Procurement of necessary basic structures for research and development

6.1 Setting up isolators to be used for studies involving vaccine testing and virus transmission in poultry 6.2 Building BSL 3 laboratory for experiments involving infected poultry and mammal species such as pigs.

6.3 Purchasing bioreactors to augment the capability to synthesize anti-influenza drugs

7. Human resource development

8. Knowledge Management

8.1 Proposal on policy and strategic planning

8.2 Knowledge management through an Explicit Knowledge approach : Obtaining problems addressed among affected farmers for prevention of future outbreaks 9. Development of information resources

9.1 Setting up database/ information resources / website related to emerging infectious diseases

9.2 Collected information on avian influenza research carried out in Thailand

9.3 Collected information on avian influenza research carried out worldwide.

10. International cooperation

10.1 Establishing international networks • Within Southeast Asia (ASEAN) • Within Asia • Global network Collaborate with world-renown research institutes e.g., St.Jude Children’s Research Hospital, USA, Harbin Veterinary Research Institute, China

10.2 Organizing international conferences participated by research institutes around the globe

11. Public communication

Activities involving public communication include: o Newspapers o Press conference o Books and other press materials o Active communication with local public health staff to promptly and effectively deliver

the knowledge to rural farmers

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Influenza research map

1.Migratory birds, wild birds, non-industrial poultry

3. Man

2.1 Intensive Industrial

Scale Farming (Broiler, Layer)

2.2 Indigenous

Poultry (fighting cocks,backyard chicken, free-range ducks

5.Outbreak

PandemicInfluenza vaccine

4.Antivirals

Surveillance

• SRRT availability & capacity

& investigation

Seasonal influenza vaccine

Transmission / Exposure

Risk Communi-

cation

AI Surveillance

•and control network Biosecurity,

Compartmentalization, movement control

& other vet

• infection monitoring • vaccine efficacy & safety • Feasibility of alternative husbandary

models • Extent of actual use of AI vaccine in

farms • Vaccine efficacy & safety • Viral shedding in vaccinated animals • Local vaccine development

• Infection rate • Transmisibility

(need biosafety lab.)

•Compart mentalization •biosecurity

• Vaccine development • Efficient SRRT

management model • Community awareness & attitude

• Pilot production • Clinical trials of

candidate vaccine • Quick test devel. & assessment • Risky practices

• Effective communi -cation model • Lab.development

• AI infection rates in migratory and local birds

• Host factors to infection, including genetics • Migration paths

• predictors of severity

• Natl. Flu vaccination policy

• Vac cost-effectiveness

• Feasibility of local production

• Pandemic impact estimation

• Pandemic control measures

• Viral genome changes

• Viral viability • Asymp.

infections in risk groups

• Oseltamivir synthesis and formulation

• Feasibility of local production • Development of alternative

antiviral, including herbal (oseltamivir/zanamivir)

• Drug Resistance Monitoring

2.Poultry

Other animals, esp mammals

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Chapter 4 Proposed Research for Collaboration

The proposed research for collaboration with neighboring countries has been proposed, extensive discussed and agreed upon by several Thai agencies including the HSRI (The Health System Research Institute), NRCT (The National Research Council of Thailand), TRF (The Thailand Research Fund), NSTDA (National Science and Technology Development Agency), DLD (Department of Livestock Development), DNP (National Park, Wildlife and Plant Conservation Department) and NHF (National Health Foundation). The research has been proposed in the following areas.

1. Surveillance and disease control in birds (NRCT/TRF/DLD/DNP) a) Migratory/ wild birds/ non-industrialized poultry b) Vaccination in non-industrialized poultry c) Networking/ capacity building d) Monitoring of genetic variation/ evolution e) Effectiveness implications of disinfectant in outbreak

2. Husbandary model and alternative model/compartmentalization (NRCT/TRF/DLD) 3. Surveillance and disease control in humans (NSTDA/NHF)

a) Asymptomatic infection b) Immune response in survivors

4. Protocol development for outbreak control at source of origin/ study

control of host factors/ treatment of infected patients a) Monitoring genetic variation/ evolution b) Risk communication

5. Development of antiviral and vaccine (NSTDA) a) Oseltamivir/ zanamivir/ herb b) Vaccine development & clinical trial c) Antiviral resistance monitoring

6. Socio-economic: operational research on public health system of country members. (NSTDA/HSRI/TRF)

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